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OBJECTIVE: Mesenchymal stem cells (MSC) are transplanted for periodontal tissue regeneration, and the periodontal ligament (PDL) is regenerated using a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells, growth factors, and amniotic membrane (AM). Dental pulp (DP)-derived cells can be easily obtained from extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells were cultured on AM as a culture substrate for immunohistochemical examination. METHODS: Wisdom teeth extracted from three adults were cut along the cement-enamel border. DP tissue was collected, minced, and primarily cultured. After three or four passage cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-E) and immunofluorescence staining. RESULTS: DP-derived cells cultured on AM formed a layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44, 105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. CONCLUSIONS: DP-derived cells proliferated on AM, while retaining the properties of DP, which allowed the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained MSC, which suggests its potential application in periodontal tissue regeneration.

Objective The usefulness of the amniotic membrane as a cell culture substrate has led to its use in the development of dental pulp‐derived cell sheets. We induced osteoblastic differentiation of dental pulp‐derived cell sheets and conducted histological and immunological examinations in addition to imaging assessments for regeneration of bone defects. Methods Dental pulp cells were obtained by primary culture of the dental pulp tissue harvested from extracted wisdom teeth. These cells were maintained for three to four passages. Subsequently, the dental pulp cells were seeded onto an amniotic membrane to produce dental pulp‐derived cell sheets. Following the induction of osteoblastic differentiation, the sheets were grafted into the subcutaneous tissue of the lower back and maxillary bone defect of a nude mouse. Histological and immunological examinations of both grafts were performed. Results Dental pulp‐derived cell sheets cultured on an osteoblast differentiation‐inducing medium demonstrated resemblance to dental pulp tissue and produced calcified tissue. Mineralization was maintained following grafting of the sheets. Regeneration of the maxillary bone defect was observed. Conclusion Induction of osteoblastic differentiation of the dental pulp‐derived cell sheets may be indicated for the regeneration of periodontal tissue.

Generation of osteoblasts from human somatic cells may be applicable in an effective transplantation therapy against bone diseases. Recently we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some transcription factor genes via retroviral vectors. However, retroviral vector-mediated transduction may potentially cause tumor formation from the infected cells, thus a non-viral gene transfection method may be more preferable for preparation of osteoblasts to be used for transplantation therapy. Here, we constructed a plasmid vector encoding Oct4, Osterix, and L-Myc that were an appropriate combination of transcription factors for this purpose. Osteoblast-like phenotypes including high alkaline phosphatase (ALP) activity, bone matrix production and osteoblast-specific gene expression were induced in normal human fibroblasts that were transfected with the plasmid followed by culturing in osteogenic medium. The plasmid-driven directly converted osteoblasts (p-dOBs) were obtained even in the absence of a xenogenic protein. The plasmid vector sequence had fallen out of the p-dOBs. The cells formed deposition of calcified bodies in situ after transplantation into mice. These results strongly suggest that p-dOBs can be put into practical use for a novel cell-based therapy against bone diseases. J. Cell. Biochem. 117: 2538-2545, 2016. © 2016 Wiley Periodicals, Inc.

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